CHAPTER 34 Techniques for Artificial Insemination of Cattle with Frozen-Thawed Semen
In the modern commercial dairy herd, management of the artificial breeding program is a major responsibility. It requires constant attention and expertise by personnel responsible for semen handling and insemination. Whether the responsibility is contracted to a professional artificial insemination (AI) technician or is assigned to employees of the farm, the requirements for success are the same. The artificial breeding program depends on the inherent fertility, health, nutrition, and management of both the cows and the bulls, as well as on the ability of the handlers to preserve, store, and deliver an adequate dose of viable semen to the cow at the proper time during the estrous cycle.
The primary objective in handling semen properly is to conserve the fertile life of sperm until deposition in the female. This is accomplished by minimizing exposure of semen to injurious conditions, which have additive effects that lower the viability and fertile life of sperm. With frozen semen, the storage temperature of liquid nitrogen stops the life processes, allowing semen to be stored indefinitely if it is maintained at very low temperatures. The critical temperature appears to be −80° C. A point of paramount importance is that although it is easy to maintain frozen semen at a safe temperature, it also is easy to destroy its quality in a few moments of carelessness.
High conception rates require proper insemination techniques. The highest-quality semen placed in the healthiest cow at the proper time before ovulation will not produce a calf if the breeding techniques are not up to par. The mechanics of passing the insemination pipette through the cervix necessitate practice and should be periodically reviewed with the aid of professionals. Sanitary methods are critical to good insemination technique. Good technique also means that each insemination is performed within a reasonable amount of time. Consistently following a step-by-step procedure will assist in achieving good conception rates.
The semen storage tank is actually a large metal vacuum-sealed liquid nitrogen refrigerator encased within an extremely efficient insulation system. With proper attention and handling, most liquid nitrogen semen storage tanks give years of trouble-free service, but all storage tanks eventually fail. The semen storage tank consists of two separate chambers. Layers of aluminum foil and specially designed paper fill the space between the two chambers. Air is removed and a partial vacuum is created in the area between the two chambers. This vacuum aids in the insulation and is the major effective property of the storage tank. A specially designed stopper plugs the tank’s neck tube, insulating liquid nitrogen and semen from outside air. The tank is not airtight, however. Because liquid nitrogen cools by slowly boiling and releasing gases, a tightly stoppered tank might explode. The specially designed plug allows nitrogen gas to safely escape from the inner chamber. Storage tanks keep semen indefinitely at −196° C as long as any liquid nitrogen is present, but a depth of at least 5 cm of liquid nitrogen should always be maintained as good insurance against possible service delays. The normal field holding time or time allowed between liquid nitrogen refills varies according to the tank model and the number of times it is opened. The liquid nitrogen level should be monitored weekly with a measuring stick.
Technical advances in design and construction have produced storage tanks with liquid nitrogen holding times of 6 to 9 months. Although semen tanks are well constructed, they are still susceptible to damage from mishandling. Damage to the liquid nitrogen tank is most likely to occur when the tank is moved, so excessive movement of the tank should be avoided. The inner chamber of the tank, which contains the liquid nitrogen, is actually suspended from the outer shell by the neck tube. Any abnormal stress on the neck tube, which can be caused by sudden jarring or any excessive swinging motion, can crack the neck tube, resulting in vacuum loss and tank failure. Failure to secure the tank in a moving vehicle can result in not only increased probability of damage to equipment but also increased risk of injury to the driver or passengers if liquid nitrogen is spilled inside the passenger compartment of the vehicle.
To ensure maximum holding time, the tank should be kept in a cool and dry location away from direct sunlight, in a clean and well-ventilated area. The tank should be located in an area where it can be observed frequently to detect excessive nitrogen evaporation and development of frost around the outside neck of the storage tank. Drafts from milk coolers and furnaces should be avoided, but ventilation should be sufficient to prevent possible user suffocation, which can be caused by too high a proportion of nitrogen gas in the air. The tank should be protected from corrosion by being elevated above concrete or wet floors. A wooden dolly equipped with wheels so that it can be moved easily is ideal for tank storage. The tank should be located where it will be safe from children and vandals or locked to secure and protect semen. Particular attention must be paid to the neck and vacuum fitting. Accumulation of frost on these fittings indicates that the vacuum insulation has been lost and liquid nitrogen has been evaporating rapidly. If frost build-up is observed, a functional tank must be located and the semen transferred to it immediately. The viability of semen in the tank should be evaluated before extensive use.
As with any well-managed business, an accurate inventory of location and quantity of the semen stored in the liquid nitrogen tank is important. This is necessary not only for accounting and audit purposes but, more important, to avoid any unwanted exposure of semen during searching for the desired sire’s semen before removal for thawing. A simple semen tank inventory log enables the exact location and quantity of semen remaining to be determined quickly.
Maintenance of low temperatures is important to the successful storage of frozen semen. In 1957, Van Demark and coworkers1 compared the motility of semen frozen to and stored at −79° C, −65° C, −51° C, −37° C, and −23° C. After 1 day of storage, the motility of semen stored at −37° C and −23° C was basically zero. Semen stored for 1 day at 79° C had 38% motility, compared with 29% and 14% motility for 65° C and 51° C storage, respectively. In studies in which −79° C storage was compared with −96° C2 or −92° C and −196° C 79° C was inferior to the lower temperatures when judged by maintenance of sperm motility as measured on thawing.
The reasons that subzero temperatures of −79° C and above cannot adequately preserve sperm are unclear and probably are quite complex. Rapatz,2 however, has proposed that rearrangement in the crystalline structure of frozen semen (recrystallization) may be one cause. This phenomenon has been observed to occur in frozen semen down to a temperature of −80° C. Below −80° C the structure of ice is more stable, and below −100° C it is very stable. Although no direct evidence exists for recrystallization, a correlation has been found between storage injury of spermatozoa and recrystallization.
In addition to the obvious error of permitting a liquid nitrogen storage tank to go dry, stored semen also may be exposed to adverse high temperatures when straws are being removed for thawing. Figure 34-1 shows the temperature gradient that exists in the neck of a typical liquid nitrogen storage tank. As may be noted, dangerously high temperatures prevail in the upper third of the neck of the tank where canes and goblets are raised for removal. When straws are exposed to these temperatures the semen temperature rises quickly. The thermal response of semen in 0.5-ml straws exposed to temperatures of −22° C (2 inches from the top of the tank) and 5° C (1 inch from the top of the tank) is shown in Figure 34-2. The time required to reach −100° C to −80° C, which is the beginning of ice recrystallization, is approximately 10 to 12 seconds for both temperatures. Thermal injury to sperm is permanent and cannot be corrected by returning semen to the liquid nitrogen. For optimal maintenance of sperm viability, canisters and canes containing semen should be raised into the neck of the tank only for the time required to retrieve a single straw. This time should not exceed 5 to 8 seconds.
(From Saacke RG: Concepts in semen packaging and use. Proceedings of the Eighth Conference on Artificial Insemination in Beef Cattle of the National Association of Animal Breeders, 1974, p 15.)
(From Saacke RG, Lineweaver JA, Aalseth EP: Procedures for handling frozen semen. Proceedings of the Twelfth Conference on Artificial Insemination in Beef Cattle of the National Association of Animal Breeders, 1977, p 49.)